Carboxyl-modified superabsorbent protein hydrogel

ABSTRACT

The present invention is a biodegradable, reversibly-swellable, polyvalent cation-binding, protein hydrogel which comprises an acylated protein matrix in which the acylated protein matrix is crosslinked with a bifunctional crosslinking reagent, and treated with a polar organic solvent, and a method of making the same.

FIELD OF THE INVENTION

[0001] The present invention relates to protein hydrogels. Morespecifically, the present invention relates to chemically modifiedprotein hydrogels which are capable of absorbing a large amount of wateror other liquid per unit mass.

BIBLIOGRAPHIC CITATIONS

[0002] Complete bibliographic citations to the numbered referencesdiscussed herein are contained in the bibliography section, directlypreceding the Claims.

DESCRIPTION OF THE RELATED ART

[0003] Beginning in the early 1970's, and continuing to the present day,there has been a growing awareness that the continued widespread use ofnon-biodegradable, petroleum-based polymeric materials may pose seriousenvironmental concerns. These concerns are heightened by productionstatistics showing the enormous and still-growing volume ofnon-biodegradable plastics produced annually, the vast majority of whichare ultimately interred in landfills. This raises concerns not only asto the amount of space available for solid waste disposal (which isdisappearing at an increasingly rapid pace), but also raises equallyserious concerns that the leaching of toxic monomers and oligomers fromlandfilled plastics will contaminate ground water, thereby causinghealth problems in humans and animals.

[0004] In addition to concerns regarding human health and theenvironment, the world-wide depletion of petroleum reserves, incombination with wildly fluctuating petroleum prices due to politicaland economic conflicts, indicates that less dependence onpetroleum-derived products might be prudent. Therefore, the developmentof alternative, and renewable, resources for industrial products isneeded.

[0005] Because of the factual and/or perceived economic, environmental,and public health concerns accompanying non-biodegradable,petroleum-based products, a non-petroleum-based, environmentally safe,biodegradable, and renewable source for industrial products is needed.As evidenced by the following references, several types of usefulproducts have been fabricated from renewable sources of startingmaterials.

[0006] For instance, Mann, U.S. Pat. No. 2,729,628, describes a processfor increasing the intrinsic viscosity of a long chain polypeptide,particularly natural proteins such as fish, peanut protein, soybeanprotein, casein, egg albumin, and blood albumin by acylating the proteinwith terephthalyl dichloride. Here, the protein is reacted with theterephthalyl dichloride using the Schotten-Baumann method at atemperature of from about 0° to 30° C.

[0007] Young et al., U.S. Pat. No. 2,923,691, describe thepolymerization of animal proteins to improve their characteristics foruse as animal glue. Young et al. introduce aldehydes to an animal glueprotein so as to modify the viscosity and jelly characteristics of theglue product without solidifying or insolubilizing the protein. Here,Young et al. are interested in increasing the viscosity and jellystrength of last run animal glues, which tend to be of inferior quality.The process described by Young et al. includes two steps: first, acyanic acid salt is reacted with the protein material; second, analdehyde, such as formaldehyde or glucose, is added to the proteinmaterial.

[0008] Two patents to Miller (U.S. Pat. Nos. 3,685,998 and 3,720,765),and assigned to the Monsanto Company, describe improved protein feedmaterials for ruminants. In the Miller patents, protein feeds arerendered resistant to digestive breakdown in the rumen, but not in theabomasum and intestines, by treating protein-containing feed materialwith a polymerized unsaturated carboxylic acid or anhydride. Forinstance, the proteinaceous feedstuff is treated with a polyanhydridesuch as poly(maleic anhydride). This renders the protein feedstuffsubstantially indigestible in the fluid medium of the rumen, yet stilldigestible in the acidic media of the abomasum and the intestines. Inthis manner the proteins of the feedstuff are spared breakdown in therumen, and are available for absorption in the subsequent digestiveorgans.

[0009] Three patent references to Battista (U.S. Pat. Nos. 4,264,493;4,349,470; and 4,416,814) describe the formation of protein hydrogelstructures formed from natural proteins having molecular weights notexceeding 100,000 by dissolving the protein in an aqueous acidicsolution, crosslinking the protein, and air drying the solution to amoisture content not exceeding 10 percent. The Battista patents arelargely drawn to the formation of clear products such as soft contactlenses, ophthalmological films, and the like.

[0010] Although Battista refers to the compositions described therein ashydrogels, that term is defined within the Battista references asmeaning “a crosslinked protein polymer of natural origin having anaverage molecular weight of about 100,000 or less, capable of beingswollen by water over a wide range of water contents ranging from as lowas 30 percent to 1,000 percent and higher while possessing usefulTheological control properties for specific end product uses.” (See forinstance, U.S. Pat. No. 4,264,493, column 1, lines 19-27.) The hydrogelsdescribed by Battista are not designed to be superabsorbent. Rather,they are designed to be optically clear and to have sufficientmechanical integrity to function as soft contact lenses.

[0011] The protein hydrogel structures described in the Battista patentsare made from natural protein raw materials that form clear solutions inwater. The protein raw material is first dissolved in an acidic aqueoussolution of from pH 3.5 to about pH 5.5. A crosslinking agent is thenadded to the acidic protein solution. Battista's preferred crosslinkingagent is Formalin (37 percent formaldehyde); however, Battista describesother suitable crosslinking agents which may be used, includingglutaraldehyde. It must be noted, however, that the Battista patents donot describe acyl-modification of the protein starting material. Nor dothe Battista patents describe a superabsorbent protein hydrogel. Theprotein hydrogels described in the Battista references are designed tohave increased wet strength capabilities, thereby enabling their use insoft contact lenses.

[0012] Many disadvantages which accompany synthetic hydrogels (such asnon-biodegradability) can be overcome by using hydrogels derived fromnatural polymer sources. In addition to chemically-crosslinked proteinhydrogels, such as those described by Battista, many proteins can bethermally induced to form gels. The most critical requirements for anytype of biopolymer hydrogel are that the gel should have the capacity toabsorb a large amount of water relative to its mass upon rehydration,and that the gel material itself should resist dissolution.

[0013] However, conventional thermally-induced protein hydrogels do notswell to their original gel volume after they have been dehydrated. Thisdecreased swelling capacity is related to increased hydrogen bonding, aswell as electrostatic and hydrophobic interactions which occur in thedehydrated protein. The loss of swelling of thermally-induced proteinhydrogels limits their range of industrial applicability.

[0014] U.S. Pat. No. 5,847,089 to Damodaran et al. describes a proteinhydrogel which is superabsorbent, reversibly swellable, biodegradable,and capable of binding cations. The protein hydrogel described inDamodaran et al. is made by treating a protein with an acylating agentand crosslinking the acylated protein with a bifunctional crosslinkingagent to form a protein hydrogel. A shortcoming of Damodaran et al. isthat residual crosslinking agent can remain in the gel, thereby makingthe gel less desirable for some applications in which residues fromcrosslinkers, such as gluteraldehyde, are a concern.

[0015] In view of this, there is a clear need for a protein hydrogelwhich is highly absorbent, biodegradable, reversibly swellable, andwhich is substantially free of residual crosslinkers used to producesuch hydrogels. The present invention provides such a protein hydrogel.

[0016] Perhaps the most desirable of renewable production materials isagricultural biomass. This is due, in large part, to the tremendousamount and variety of agricultural products which are produced in theUnited States. For instance, biomass (mainly maize) is currently used toproduce ethanol for fuel. Fibrous biomass is widely used in the paperand forest products industry. Starch-derived products are also widelyutilized in various industrial applications, such as the packingindustry, in addition to their use in the food industry.

[0017] However, among biopolymers, proteins are perhaps the mostunder-utilized and under-rated in terms of their industrialapplications. They are primarily regarded solely as functional andnutritional ingredients in foodstuffs. Their enormous potential asstructural elements in non-food industrial applications is largelyunrecognized and unrealized. This is unfortunate because proteins offerseveral distinct advantages over more conventional types of biomass.

[0018] For example, unlike polyol-based natural polymers, such ascellulose and other carbohydrates, proteins contain several reactiveside groups, including amino, hydroxyl, sulfhydryl, phenolic, andcarboxyl moieties. These reactive groups can be used as sites ofchemical modification and crosslinking to produce novel polymericstructures. The present invention relates to such a novel polymericstructure: a protein-based, biodegradable, superabsorbent hydrogel.

[0019] As a generic class of polymers, hydrogels of all types find highvolume uses in industrial applications, consumer products, andenvironmental applications. Such applications include diapers,catamenial devices, and industrial absorbents. As used herein, theunqualified term “hydrogel” refers to any naturally-occurring orsynthetic material which exhibits the ability to swell in water or someother liquid and to retain a significant fraction of liquid within itsstructure, but which will not dissolve in the liquid.

[0020] Several synthetic hydrogel materials are currently in use. Theseinclude such synthetic hydrogels as poly(hydroxyalkyl methacrylates),polyacrylate, poly(acrylamide), poly(methacrylamide) and derivativesthereof, poly(N-vinyl-2-pyrrolidone), and poly(vinylalcohol). Whilethese synthetic hydrogel polymers exhibit several interestingproperties, their use in industrial, consumer, and environmentalapplications is less than desirable because of the toxicity of residualmonomers and oligomers which are normally present in these gels.Moreover, the poor biodegradability of these synthetic hydrogels alsoposes the long-term environmental concerns discussed above.

[0021] Clearly then, there exists the need for a biodegradable,superabsorbent, biomass-derived hydrogel which exhibits reversibleswelling, and which is substantially free of residual crosslinker usedto produce such hydrogels.

SUMMARY OF THE INVENTION

[0022] In view of the above discussion, it is a principal aim of thepresent invention to provide a protein hydrogel which is superabsorbent,reversibly swellable, biodegradable, and capable of binding divalentcations. The protein hydrogel is also substantially free of residualcrosslinker used to produce such hydrogels.

[0023] A further aim of the invention is to provide a protein hydrogelwhich can be formed from a wide range of protein starting materials, andwhich can be used as a substitute for wholly synthetic hydrogels.

[0024] In its simplest embodiment, the present invention relates to aprotein hydrogel which comprises an acylated protein matrix which hasbeen crosslinked with a bifunctional crosslinking reagent, and which hasbeen treated with a polar organic solvent to remove residualcrosslinker.

[0025] More specifically, the present invention includes a proteinhydrogel which comprises a fish protein isolate which has been acylatedby treatment with ethylenediaminetetraacetic acid dianhydride (EDTAD) toyield an acylated protein matrix. The acylated protein matrix is thencrosslinked with glutaraldehyde to yield a biodegradable,superabsorbent, protein hydrogel. The crosslinked gel is then treatedwith a polar organic solvent (preferably ethanol).

[0026] The solvent treatment induces conformational reorganization inprotein chains in the gel network, which apparently increasesflexibility and hence the rate and extent of relaxation of the polymernetwork as water diffuses into the network. In addition to improving theswelling properties, the ethanol treatment offers the followingadvantages: 1) ethanol dehydrates the gel and thereby eliminates theneed for drying the gel; 2) ethanol extracts low molecular weightoff-odor compounds from the protein gel, especially from the fishprotein gel, and thereby improves its acceptability in several consumerproducts-the ethanol-treated fish protein hydrogel was found to becompletely free of fishy off-odor compared to that made without ethanoltreatment; and 3) ethanol also extracts any residual un-reactedglutaraldehye (believed to be carcinogenic) that might be present in thegel.

[0027] The protein hydrogels of the present invention are capable ofabsorbing more than 100 times (and often more than 200 times) their dryweight in water. They are also capable of sequestering divalent cations.

[0028] The present invention also includes a method of making theprotein hydrogel described immediately above. The method includes thesteps of treating a protein with an acylating agent to yield an acylatedprotein matrix, crosslinking the acylated protein matrix with abifunctional crosslinking agent, and treating the crosslinked matrixwith a polar organic solvent (preferably ethanol) to yield the proteinhydrogel.

[0029] In more detail, the present invention includes a method of makinga protein hydrogel which includes the steps of dissociating and/orunfolding protein molecules within an aqueous protein solution byapplication of heat, and then adding an acylating agent to the proteinsolution to yield an acylated protein. The acylated protein is thencrosslinked by addition of a bifunctional crosslinking agent. Thecrosslinked matrix is then treated with a polar organic solvent(preferably ethanol) to induce conformational reorganization in proteinchains in the gel network to yield the protein hydrogel.

[0030] The present invention is a protein hydrogel having theabove-described properties. The protein from which the protein hydrogelis derived can be from any plant or animal source, without limitation. Apreferred protein source, its preference derived in large part from itsabundance and low cost, is fish-derived protein.

[0031] The protein hydrogel of the present invention is made by firstchemically modifying lysyl residues of a protein by the addition of oneor more carboxyl moieties thereto. Preferably, this is done by acylationof the lysyl residues with a polycarboxylic acid anhydride. This isfollowed by crosslinking of the protein chains with a bifunctionalcrosslinking agent and treating the protein with a polar solvent toyield a protein hydrogel that exhibits superabsorbent, pH-sensitive andionic strength-sensitive reversible swelling, and which is substantiallyfree of residual crosslinker.

[0032] The protein hydrogel of the present invention also strongly bindsdivalent cations. This enables the protein hydrogel to function as acationic sequestering agent. The protein hydrogel can be used to removedivalent metal cations and organic cations from ground water, effluentliquid waste streams, and the like.

[0033] In operation, the protein hydrogel can be used wherever highabsorption of liquid, or sequestering of divalent cations is desired.Potential end uses for the protein hydrogel include cosmetic products,diapers, tampons and menstrual pads, industrial absorbents, spill damsand sealers, ground and waste water reclamation applications, heavymetal sequestration, and the like.

[0034] The objects and advantages of the invention will appear morefully from the following detailed description of the preferredembodiment of the invention made in conjunction with the accompanyingdrawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035]FIG. 1 is a graph showing water uptake of unmodified fish protein(FP) (Δ) and 80% EDTAD-modified FP hydrogels (∘).

[0036]FIG. 2 is a graph illustrating the rate of water uptake of thegels of ethanol-treated unmodified-FP (Δ) and ethanol-treated 80%EDTAD-modified FP (∘).

[0037]FIG. 3 is a graph depicting the effect of ethanol treatment onwater uptake of 60% EDTAD-modified soya protein (SP) (Δ) andethanol-treated 60% EDTAD-modified soya protein (∘).

[0038]FIG. 4 is a graph showing far UV-CD spectra of swollen gels of 80% EDTAD-FP prepared with the ethanol treatment (bold line) and withoutthe ethanol treatment (thin line).

[0039]FIG. 5A is a graph illustrating the effect of ethanol treatmentswelling properties of 80% EDTAD-FP in 0.1M NaCl at 36° C.(Δ=EDTAD-modified FP; ∘=ethanol-treated EDTAD-modified FP).

[0040]FIG. 5B is a graph depicting the effect of ethanol treatmentswelling properties of 80% EDTAD-FP in 0.15 M NaCl at 36° C.(Δ=EDTAD-modified FP; ∘=ethanol-treated EDTAD-modified FP).

[0041]FIG. 6 shows Van't Hoff plots of log M_(s) (maximum swelling) vs.1/T for 80% EDTAD-modified FP (Δ) and ethanol-treated EDTAD-modified FP(∘), showing the effect of temperature on equilibrium water uptake.

DETAILED DESCRIPTION OF THE INVENTION

[0042] At the heart of the present invention is chemical modification ofa protein so as first to introduce carboxyl moieties into then-butylamino side groups of lysine residues within the protein. Themodified protein molecules are then crosslinked using a bifunctionalcrosslinking agent to yield a protein hydrogel. The crosslinked proteinmatrix is then treated with a polar organic solvent to remove residualcrosslinker and to yield a biodegradable, superabsorbent proteinhydrogel which is substantially free of residual crosslinker.

[0043] The swelling properties of protein hydrogels described in U.S.Pat. No. 5,847,089 to Damodaran et al. can be dramatically improved bytreating the cross-linked gel with ethanol. While not being limited to aparticular mode or mechanism of action, this improvement occursapparently because of ethanol-induced conformational reorganization inprotein chains in the gel network. This rearrangement apparentlyincreases flexibility and hence the rate and extent of relaxation of thepolymer network as water diffuses into the network. In addition toimproving the swelling properties, the ethanol treatment providesseveral additional advantages:

[0044] Treating the crosslinked protein matrix with a polar organicsolvent dehydrates the gel. This eliminates the need for drying the gelafter it has been formed.

[0045] Treating the crosslinked polymer matrix also extracts lowmolecular weight, off-odor compounds from the protein gel. This isparticularly beneficial when the gel is formed from fish protein (oranimal protein), which tend to retain an off odor. This effect greatlyimproves the commercial acceptability of the gel for use in numerousconsumer products. Ethanol-treated fish protein hydrogel according tothe present invention was found to be completely free of fishy off-odorcompared to fish protein hydrogel made without ethanol treatment.

[0046] Treatment with a polar organic solvent extracts any residualun-reacted crosslinking reagent from the gel. This is especiallybeneficial if glutaraldehye (which is suspected to be carcinogenic) isused as the crosslinker. Treatment with the polar organic solventremoves any residual glutaraldehyde.

[0047] As noted above, the protein starting material can be selectedfrom any source, animal, vegetable, or microbial, without limitation.For instance, while fish protein is preferred due to its low cost, theprotein hydrogel described herein can be manufactured from other oilseedproteins, leaf proteins (e.g., alfalfa), microbial proteins, animalproteins, and proteins recovered from food processing wastes. Crudeprotein concentrates, as well as protein isolates will function equallywell in the present invention. And, since the protein hydrogel is notgenerally intended for consumption, the starting material need not be offood grade.

[0048] The preferred protein source is fish, which are extracted withwater to yield a fish protein isolate. Isolation of crude fish protein(FP) from fresh fish is carried out in conventional fashion, asdescribed elsewhere (1). Generally, fresh fish upon arrival arefilleted, chopped and blended with chilled de-ionized water at ameat-to-water ratio of 1:9. The suspension is then adjusted to pH 12 andstirred for 30 min. The suspension is filtered to remove the insolubleparticles, and the filtrate is dialyzed against water and lyophilized.Polar organic solvents useful in the present invention include C₁-C₄alcohols (preferred), C₁-C₄ ketones (acetone preferred), and C₁-C₄aldehydes (less preferred due to odor). Particularly preferred solventsfor use in the present invention are ethanol, acetone, propanol, andbutanol. The most preferred polar organic solvent is ethanol which willbe used to exemplify the claimed invention. (The limitation to adescription of using ethanol in the invention is for brevity only.)

[0049] Further isolation of the fish protein can be accomplished in anysuitable fashion. For instance, this can be accomplished by treatmentwith acid to precipitate the proteins dissolved within the extract toyield fish protein isolate (FPI). The protein will normally precipitatefrom solution at about pH 4.5 The FPI may optionally be dialyzed orfurther purified (e.g., by recrystallization) if desired. Otherisolation methods, such as evaporation of the solvent, orchromatography, can be used with equal success. Again, while the presentinvention may be practiced with any type of protein, for brevity andclarity only, the remainder of the specification shall be limited to asdescription of protein hydrogels made using the above-described FPI(which is the preferred protein).

[0050] After isolation, the FPI is then modified with a carboxylicgroup-containing acylating agent. The acylating agent reacts withn-butylamino groups of lysine residues within the FPI, and functions tointroduce carboxyl moieties into the FPI. Preferably, the acylatingagent is a polycarboxylic anhydride, a mono-anhydride, a dianhydride, ora combination thereof. As used herein, the term “anhydride” shall meanany of the preceding types of anhydrides. Suitable dianhydrides whichcan be used in the present invention include, for example,benzenetetracarboxylic dianhydride, cyclobutane tetracarboxylicdianhydride, diethylene-triamine-pentaacetic dianhydride, andethylenediaminetetraacetic acid dianhydride (EDTAD). EDTAD is thepreferred acylating agent. Again, for brevity, the description whichfollows will be limited to addition of EDTAD to the FPI. This is forbrevity and clarity only, and is understood not to limit the inventionclaimed herein in any fashion.

[0051] Introduction of the EDTAD into the FPI is performed by step-wiseaddition of solid EDTAD to an aqueous solution of FPI. It must beremembered, however, that EDTAD is a bifunctional reagent which iscapable of cross-linking polypeptides either inter- or intramolecularly.Two possible reaction pathways for the reaction of EDTAD with a proteinare shown below, wherein PRO is the protein being modified:

[0052] In Reaction I, one molecule of EDTAD reacts with two lysylresidues to form a linkage. When the reaction of the protein with EDTADproceeds by Reaction I, the result is the incorporation of only onecarboxyl moiety per lysyl residue. Moreover, if Reaction I occursbetween subunits of a protein molecule, the intramolecular crosslinkingmay impair swelling of the modified protein.

[0053] In Reaction II, one molecule of EDTAD reacts with one lysylresidue and one water molecule. In this reaction, three carboxylmoieties per lysyl residue are incorporated into the protein, and nolinkages are formed. This greatly increases the net anionic charge ofthe modified protein, which aids in unfolding the protein structure.Because no linkages are formed, the swellability of the modified proteinis not impaired.

[0054] In light of the bifunctionality of EDTAD, in order to form aprotein hydrogel having maximum absorbency, EDTAD should be added to theFPI under conditions which favor Reaction II over Reaction I. Conditionswhich favor Reaction II over Reaction I are those conditions wherein theprotein is present in dilute solution, and the individual proteinmolecules are partially dissociated and/or denatured, thereby lesseningthe possibility of EDTAD reacting with two protein molecules. Thereaction can be carried out at a temperature range of from about 5° C.to about 100° C. It is preferred that the reaction be conducted atmildly elevated temperatures, from ambient to about 100° C, under basicconditions, about pH 8 to pH 12. However, the reaction conditions shouldnot be so rigorous as to cause hydrolytic degradation of the proteinchains.

[0055] The preferred reaction protocol to optimize reaction of theprotein by the mechanism of Reaction II is to first incubate the FPI ina dilute aqueous solution of about pH 12, at a mildly elevatedtemperature of about 65° C. The concentration of FPI in the solutionshould be on the order of about 1 percent. The FPI should be incubatedfor approximately 30 minutes at 65° C. Longer incubations times areacceptable so long as alkaline hydrolysis does not occur. The incubationperiod serves to dissociate and/or denature the protein molecules of theFPI.

[0056] The incubation may also be performed under acidic conditions,down to about pH 2. However, since the preferred acylation reactiontakes place in alkaline solution, it is preferred that the incubation isalso done under alkaline conditions so as to minimize salt formationduring the acylation reaction.

[0057] After incubation, the solution is cooled to room temperature anda calculated amount of EDTAD is added in incremental amounts withcontinuous stirring. After complete addition of EDTAD, the reactionmixture is stirred constantly for 3 h while maintaining the pH at 12.0,preferably by the addition of base (preferably NaOH). This can be doneautomatically using a commercially-available pH-Stat apparatus (FisherScientific). Under these conditions, little or no alkaline hydrolysis ofthe FPI occurs. At the end of the reaction, the pH of the proteinsolution is then adjusted 4.5 to precipitate the protein. The suspensionis centrifuged at 10000 ×g for 15 min. The protein sediment is washedwith water at pH 4.5 and centrifuged. The final protein precipitate isthen re-dissolved in water at pH 7.0 and lyophilized. The extent ofacylation, i.e., the percentage of lysyl residues modified with EDTAD,is determined by the trinitrobenzenesulfonic acid (TNBS) method (2).

[0058] The extent of acylation can be varied so as to modulate thephysical characteristics of the final gel product. This can be donequite easily by varying the ratio of protein to added EDTAD (or otheracylating agent). The greater the amount of EDTAD added per unitprotein, the greater the extent of modification.

[0059] Of course, the reaction conditions can be easily adjusted by oneof skill in the art such that Reaction I prevails. For instance, ashortened incubation period, or omission of the incubation step entirelywill tend to favor Reaction I, as will adding the EDTAD to a moreconcentrated FPI solution. Performing the acylation at an alkalinitycloser to pH 9 tends to favor the Reaction I pathway over the ReactionII pathway.

[0060] Adjusting the relative rates of the two reactions will change thecharacteristics of the final protein hydrogel. While optimization ofReaction II yields a protein hydrogel having superior swellability andgreater overall anionic charge, optimization of Reaction I yields astiffer, less absorbent hydrogel, which is desirable in someapplications. Knowledge of the interplay between the two reactionsallows the physical characteristics of the final gel product to betailored to fit a wide variety of final applications.

[0061] The ratio of reaction by the Reaction I pathway versus theReaction II pathway can be determined by electrometric titration ofvarious modified and unmodified FPI samples. The titration curves of themodified samples are then compared to unmodified samples subjected tothe same reaction conditions. The number of carboxyl groups per 10⁵gmole of protein is calculated from the number of moles of H+ iondissociated (or, by the number of moles of NaOH consumed) by the proteinduring titration from pH 2.0 to the isoionic point of the protein.Titration curves for native soy protein, and soy protein subjected to pH12 and 65° C. are essentially identical (data not shown), illustratingthat heat treatment at pH 12 does not result in deamidation of theglutamine and asparagine residues of soy protein. Presumably, FPIbehaves similarly. Knowing this, any increase in the carboxyl groupcontent of FPI modified under these conditions must be due toincorporation of EDTAD at the lysyl residues of the FPI.

[0062] It must also be remembered that the crosslinking step, describedin full below, also utilizes lysine residues within the protein tocrosslink the protein chains. Therefore, it is preferred that the extentof modification not exceed 90% of available lysine residues. Thismaximum extent of modification should also be decreased if the startingprotein is particularly low in lysine residues. In order to obtain theadvantages of increased carboxyl moiety content, it is preferred that aminimum of 50% of the lysine residues of the starting protein beacylated.

[0063] After acylation, the protein solution is exhaustively dialyzedagainst deionized water to remove salts (in this case, primarily sodiumEDTA) formed in the reaction. The dialyzed modified protein may beoptionally lyophilized to yield an acylated protein. Alternatively, theprotein can be precipitated by lowering the pH to 4.5, followed bycentrifugation. The protein precipitate can then be dissolved in waterand pH8to9.

[0064] EDTAD is the preferred acylating agent because, inter alia, it isessentially non-toxic. The only reactive groups introduced into theprotein by the addition of EDTAD are the carboxyl groups. When added tothe protein isolate according to the protocol described above, anyunreacted EDTAD will readily react with water and NaOH, to be convertedinto sodium ethylenediaminetetraacetic acid (EDTA). Since sodium EDTA isa “Generally Regarded As Safe” (GRAS) food additive, there is no concernin regard to the toxicity or environmental safety of any residual amountof sodium EDTA (if any) remaining in the modified protein. Unlikepoly(acrylate) or poly(acrylamide)-based hydrogels, which may containresidual monomers which are toxic, the present protein hydrogel, if itcontains any residual reagents, would only contain residual sodium EDTA.

[0065] While not being limited to any particular mode of operation, itis believed that the EDTAD acylating agent, by reaction with the lysylresidues of the protein, causes extensive unfolding of the proteinmolecules via intramolecular electrostatic repulsion caused by thecarboxylic acid substituents on the acylating agent. This is believed toconvert the rigid, globular structure of fish globulins into arandom-coil-type, polyanionic polymer. The substantial polyanioniccharacter which the carboxylic acid moieties impart to the proteinisolate are believed to provide numerous sites for water binding.

[0066] After acylation, the dialyzed and optionally lyophilized modifiedprotein isolate is crosslinked using a bifunctional crosslinkingreagent. A wide variety of suitable bifunctional crosslinking agents areknown in the art. Dialdehydes, for instance, like dianhydrides, willalso react with lysine residues to form crosslinks between polypeptidechains. Bifunctional aldehydes are excellent crosslinking reagents. Inthe present invention, any type of dialdehyde, without limitation, canfunction as a crosslinking reagent. The preferred bifunctionalcrosslinking reagent is a bifunctional aldehyde having the formula

OCH—(CH2)_(x)—CHO

[0067] wherein X is an integer of from 2 to 8. The preferredbifunctional aldehyde from within this small group of homologs isglutaraldehyde (X=3).

[0068] Crosslinking is preferably carried out in aqueous solution. Here,in order to maximize crosslinking (both intra and intermolecularlinkages), a relatively concentrated protein solution is used, and thepH maintained at about pH 7 to pH 10. For instance, to a 10% aqueoussolution of acylated FPI at pH 9.0 is added a suitable amount of a 25%aqueous solution of glutaraldehyde. For example, about 150 μl of the 25%glutaraldehyde solution would be added to 10 ml of the 10% proteinsolution.

[0069] The mixture is then thoroughly stirred, and cured overnight atroom temperature. The cured gel is then air dried in an oven at 40° C.

[0070]FIG. 1 shows water uptake of unmodified fish protein (FP) and 80%EDTAD-FP hydrogels. The equilibrium water uptake of the unmodified FPwas only about 6 g/g gel, whereas the water uptake of the 80% EDTAD-FPhydrogel reached an equilibrium value of about 200 g/g after 24 h ofswelling. Clearly, introduction of three carboxyl groups at each lysylresidue in the protein enabled the protein network to imbibe a largeamount of water. The rate and extent of swelling of hydrogels aregoverned by the rate of diffusion of water into the gel and the rate andextent of relaxation of the polymer network in response to waterdiffusion (5-7). The data in FIG. 1 show that the rate of water uptakeby the dry (glassy) gel increased rapidly during the first hour andslowed thereafter. The initial rapid phase might be related to diffusionof water into and hydration of the charged groups in the polymernetwork. During this phase, in addition to hydrating the ionic groups,water may tend to disrupt polar protein-protein interactions in the gelnetwork. This should enhance the relaxation rate of the polymer network.However, the decrease in the rate of swelling of the hydrogel after thefirst hour indicates that although the protein was denatured by exposingit at pH 12 and 65° C. prior to crosslinking with glutaraldehyde, itsrate of structural relaxation in the gel network does not seem to becomparable to a truly random-coil polymer. Previously, (1, 4) it hasbeen shown that even after exposure of soy and fish proteins to theabove denaturing conditions, the proteins regained a significant amountof α-helix and β-sheet structures when the conditions were reversed backto pH 9 and room temperature. These folded secondary structures in thecross-linked protein network might oppose relaxation of the gel networkas water diffuses into the network. It is probable that if proteinchains in a crosslinked protein network are subjected to denaturingconditions, they may remain in a disordered state when the denaturant isremoved because of steric constraints imposed by the cross-links.

[0071] To elucidate this hypothesis, after crosslinking withglutaraldehyde (and before drying), the gel was suspended in ethanol.Due to osmosis, ethanol penetrated into the gel and water diffused outof the gel into the ethanol solvent. After 3 h of exposure, the gel lostmost of its water and collapsed into a dry solid. The dry gel wasremoved by filtration, dried in an oven at 35° C. for few minutes toremove ethanol, and its swelling properties were studied.

[0072]FIG. 2 shows the rate of water uptake of the gels of unmodified-FPand 80% EDTAD-FP prepared with the ethanol treatment. In the case ofunmodified-FP, the equilibrium water uptake was about 15 g/g, which isat least 2-fold greater than without the ethanol treatment (FIG. 1).Similarly, the equilibrium water uptake of the 80% EDTAD-FP was 425 g/g,which is more than 2-fold greater than that without the ethanoltreatment (FIG. 1).

[0073]FIG. 3 shows the effect of ethanol treatment on water uptake of60% EDTAD modified soy protein (60% EDTAD-SP). As in the case of fishproteins, ethanol treatment of soy protein hydrogel also significantlyincreased its rate and extent of swelling. This indicates that theswelling properties of all protein-based hydrogels can be dramaticallyimproved by treating the freshly crosslinked gel with ethanol.

[0074] Comparison of the data in FIGS. 1 and 2 suggest that both theinitial rate and the extent of swelling of the gels are markedlyimproved by the ethanol treatment. This is presumably due toethanol-induced denaturation of protein in the gel network. To determineif ethanol treatment causes structural changes in proteins in the gelnetwork, the CD spectra of swollen gels were analyzed.

[0075]FIG. 4 shows far UV-CD spectra of swollen gels of 80% EDTAD-FPprepared with and without the ethanol treatment. Qualitatively, the CDspectrum of the gel which was not subjected to ethanol treatment showeda major negative trough at 230 nm and a positive peak at 200 nm. Thistype of CD spectrum has been ascribed to proteins rich in type-I β-turns(8-11). In contrast, the CD spectrum of the ethanol-treated gel showstwo major negative troughs at 209-210 nm and 221-223 nm regions, whichare typical of a helical structure. Although it is difficult toquantitatively interpret the relationship between water uptakeproperties and the CD spectra of swollen gels, the data in FIG. 4 dohighlight the fact that ethanol treatment alters conformationalproperties of proteins in the gel network and this in turn significantlyimpacts the water uptake properties of the gels.

[0076] The effect of propanol, butanol, and acetone treatment on theswelling properties of 80% EDTAD-FP gels was also investigated. Theextent of equilibrium swelling of the gel treated with these solventswas slightly lower than gels treated with ethanol.

[0077]FIG. 5A shows the effect of ethanol treatment on the swellingproperties of 80% EDTAD-FP in 0.1M NaCl at 36° C. The saline uptake ofthe gel which was not treated with ethanol was about 24 g/g, whereasthat of the gel treated with ethanol was about 35 g/g. The rate ofsaline uptake also apparently was higher with the ethanol treated gelthan that without ethanol treatment. Similar behavior is also observedwith uptake of 0.15M saline (FIG. 5B). The improvement in the rate andextent of saline uptake of the ethanol treated gel must be related to anincrease in the rate and extent of relaxation of the protein chains inthe network.

[0078]FIG. 6 shows the effect of temperature on equilibrium water uptakeby 80% EDTAD-FP gels. The logarithm of equilibrium water uptake versusreciprocal temperature plots for both the ethanol-treated andethanol-untreated gels showed a linear behavior in the temperature range5-40° C. The slopes of these plots were the same, suggesting that theenthalpy change (ΔH) for water uptake is the same for both these gels.Thus, the net difference in the absolute amount of water uptake at anygiven temperature must arise from differences in structural flexibilityof the network (i.e., entropy related).

[0079] The following protocols are provided for illustrative purposesonly to aid in a complete understanding of the claimed invention. It isunderstood that the examples do not limit the invention claimed hereinin any manner.

[0080] Materials

[0081] Walleyed pike (fish) was obtained fresh from a local fish farm.Ethylenediaminetetraacetic dianhydride (EDTAD) and butanol were fromAldrich Chemical Co. (Milwaukee, Wis.). Glutaraldehyde (50% aqueoussolution) and propanol were obtained from Sigma Chemical Co.(St. Louis,Mo.). Absolute ethyl alcohol was purchased from Apper Alcohol andChemical Co. (Shelbyville, Ky.). Heat sealable, water wettable paper wasprocured Bolmet Inc. (Dayville, Conn.). Dialysis tubing (m.w. cut off6000-8000), acetone and ether were obtained from Fisher Scientific(Pittsburgh, Pa.). All other chemicals were of analytical grade.Deionized water was used for the swelling studies.

[0082] Protein determination

[0083] Because the modifying groups used in this study interfered withall calorimetric methods for determination of protein concentration, theprotein concentration was determined by the dry weight method (1). Aweighed aliquot of a protein stock solution in deionized water was driedto constant weight at 105° C. in a vacuum oven. The proteinconcentration was expressed as % w/v.

[0084] Modification of fish protein

[0085] Chemical modification of the lysyl residues of the protein withEDTAD was carried out as reported elsewhere (1). One percent proteinsolution in water was prepared at pH 12 and incubated for 30 min at 65°C. The solution was cooled to room temperature and a calculated amountof EDTAD was added in incremental amounts with continuous stirring.After complete addition of EDTAD, the reaction mixture was stirredconstantly for 3 h while maintaining the pH at 12.0. At the end of thereaction, the pH of the protein solution was adjusted to 4.5 toprecipitate the protein. The suspension was centrifuged at 10,000 g for15 min. The protein sediment was washed with water at pH 4.5 andcentrifuged. The final protein precipitate was then re-dissolved inwater at pH 7.0 and lyophilized. The extent of acylation, i.e., thepercentage of lysyl residues modified with EDTAD, was determined by thetrinitrobenzenesulfonic acid (TNBS) method (2).

[0086] Preparation of crosslinked hydrogel

[0087] A 10% dispersion of the EDTAD modified fish protein was preparedby dissolving the required amounts of protein in deionized water at pH10 and mixed homogeneously with an egg beater for 15 to 20 min. Becauseof high viscosity, the 10% protein dispersion looked like a thick paste.To this was added a known amount of 50% glutaraldehyde solution (whichwas also preadjusted to pH 10) so that the ratio of protein toglutaraldehyde in the final mixture was about 1:0.035 (wt/wt). Themixture was mixed uniformly for about 15 min using an egg beater andallowed to cure overnight at room temperature. The cured gel was dividedinto two equal parts. One part was dried in an oven at 40° C. The otherportion was suspended in ethanol for 3 h, during which time ethanol waschanged at least twice. The ethanol treatment caused both denaturationof protein and dehydration of the crosslinked gel. At the end of theethanol treatment, the gel was in the form of dried particles. Theparticles were further dried in an oven at 40° C. for two hours toremove ethanol and any residual moisture. Unmodified fish proteincontrol gels were prepared in the same manner. After complete drying thegels were ground to particle size less than 1.0 mm and used for swellingstudies.

[0088] Swelling kinetics

[0089] Swelling studies for all the above gels were done gravimetricallyat 36° C. A weighed amount of dried gel was taken in triplicates inheat-sealable pouches and allowed to swell in deionized water. Atspecific time intervals the bags were removed and centrifuged at 214×gin a clinical centrifuge equipped with sample holders containing plasticwire mesh for proper drainage of the expelled water to the bottom of theholder. The weight of the swollen gel was determined immediately.Appropriate controls for the wet weight of the pouch were included. Thewet pouch with swollen gel was dried in a oven at 104° C. to constantweight. The final dry weight of the gel was determined by subtractingthe dry weight of an equivalent empty pouch treated in the same manner.The water uptake was determined as g water absorbed per g dry gel. Theeffect of ionic strength on water uptake was studied in a manner similarto that described above by immersing the gel samples in 0.1M and 0.15MNaCl solutions. The influence of temperature on water uptake was studiedin the range of 5-40° C. in temperature controlled water baths.

[0090] Circular dichroic measurments

[0091] Qualitative CD measurements were made in a computerizedspectropolarimeter (On-Line Instruments Systems, Inc., Jefferson, Ga.).The gels, swollen in water, were placed between two quartz plates(2.5×2.5 cm) separated by 0.8 mm thick spacers and the cell sealed. Thefar UV CD spectrum of the sandwiched gel was recorded in the 190-240 nmrange. Twenty scans of each sample were averaged and all spectra werecorrected for the appropriate water baseline. Since the samples were inthe form of gel, the spectra were recorded in the millidegree mode,instead of the ellipticity mode which would require the exactconcentration of protein in the gel.

[0092] The lysine content of the crude protein isolated from the fishmuscle contained about 9 residues per 10,000 molecular weight. Reactionof the crude protein with EDTAD at a protein-to-EDTAD weight ratio of1:0.2 resulted in acylation of about 80% of the lysyl residues in thefish protein (80% MFP). At a protein-to-EDTAD ratio of 1:0.25 (w/w),about 90% of the lysyl residues were acylated (90% MFP). Previously, ithas been reported that, under the reaction conditions used in thisstudy, reaction of EDTAD with the protein lysyl groups results inintroduction of about 3 carboxyl groups for each lysyl residue modified(3, 4).

[0093] It is understood that the present invention is not limited to theparticular embodiment, reagents, steps, or methods described herein, butembraces all such forms thereof as come within the scope of the attachedclaims.

BIBLIOGRAPHY

[0094] 1. D-C. Hwang and S. Damodaran, Synthesis and Properties of FishProtein-Based Hydrogel. J. Amer. Oil Chem. Soci. 74, 1165 (1997).

[0095] 2. R. J. Hall, N. Trinder and D. I. Givens, Observations on theUse of 2,4,6-trinitrobenzenesulphonic Acid for the Determination ofAvailable Lysine in Animal Protein Concentrates. Analyst, 98, 673(1973).

[0096] 3. D-C. Hwang and S. Damodaran, Chemical Modification Strategiesfor Synthesis of Protein-based Hydrogel. J. Agric. Food Chem. 44, 751(1996).

[0097] 4. D-C. Hwang and S. Damodaran, Equilibrium Swelling Propertiesof a Novel Ethylenediaminetetraacetic Dianhydride (EDTAD)-modified soyprotein hydrogel. J. Appl. Polym. Sci., 62, 1285 (1996).

[0098] 5. S. H. Gehrke, Equilibrium Swelling, Kinetics, Permeability andApplications of Environmentally Responsive Gels. In Advances in PolymerScience 110—Responsive Gels: Volume Transitions II, K. Dusek, Ed.,Springer-Verlag, Berlin, Heidelberg, 1993, p. 83.

[0099] 6. Y. Okuyama, R. Yoshida, K. Sakai, T. Okano, and Y. Sakurai, J.Biomater. Sci. Polym. Ed., 4, 545 (1993).

[0100] 7. A. Dave, U. Vaishnav, R. Desai, A. Shah, B. Ankleshwaria, andM. Mehta, J. Appl. Polym. Sci. 58, 853 (1995).

[0101] 8. S. M. Kelly and N. C. Price, Biochemica Biophysica Acta 1338,161 (1997).

[0102] 9. R. W. Woody, in Methods in Enzymology, C. H. W. Hirs, Ed.,Academic, New York, 1995 vol.246, p.34-71.

[0103] 10. N. J. Greenfield, Anal. Biochem. 235, 1 (1996).

[0104] 11. J. T. Yang, C. -S. C. Wu, H. M. Martinez, in Methods inEnzymology, C. H. W. Hirs, Ed., Academic, New York, 1986 vol. 130,p.208-269.

What is claimed is:
 1. A protein hydrogel comprising an acylated protein matrix, the acylated protein matrix being crosslinked with a bifunctional crosslinking reagent to yield a crosslinked protein matrix, the crosslinked protein matrix then being treated with a polar organic solvent.
 2. The protein hydrogel according to claim 1, wherein the bifunctional crosslinking agent is a bifunctional aldehyde.
 3. The protein hydrogel according to claim 2, wherein the bifunctional aldehyde is selected from the group consisting of OCH—(CH2)_(x)—CHO wherein X is an integer of from 2 to
 8. 4. The protein hydrogel according to claim 1, wherein the bifunctional crosslinking agent is glutaraldehyde.
 5. The protein hydrogel according to claim 1, wherein the acylated protein matrix comprises a protein derived from biomass.
 6. The protein hydrogel according to claim 1, wherein the acylated protein matrix comprises a protein concentrate derived from biomass.
 7. The protein hydrogel according to claim 1, wherein the acylated protein matrix comprises a protein isolate derived from biomass.
 8. The protein hydrogel according to claim 7, wherein the protein isolate is a fish protein isolate.
 9. The protein hydrogel according to claim 1, wherein the acylated protein matrix is a protein treated with an acylating agent.
 10. The protein hydrogel according to claim 9, wherein the acylating agent is an anhydride.
 11. The protein hydrogel according to claim 10, wherein the acylating agent is a tetracarboxylic acid dianhydride.
 12. The protein hydrogel according to claim 10, wherein the acylating agent is ethylenediaminetetraacetic acid dianhydride.
 13. The protein hydrogel according to claim 1, wherein the polar organic solvent is selected from the group consisting of C₁-C₄ alcohols, C₁-C₄ ketones, and C₁-C₄ aldehydes.
 14. The protein hydrogel according to claim 13, wherein the polar organic solvent is selected from the group consisting of ethanol, propanol, butanol, and acetone.
 15. The protein hydrogel according to claim 14, wherein the polar organic solvent comprises ethanol.
 16. A protein hydrogel comprising: a fish protein isolate, the fish protein isolate being acylated by treatment with ethylenediaminetetraacetic acid dianhydride to yield an acylated protein; the acylated protein being crosslinked with glutaraldehyde to yield a crosslinked protein matrix; and the crosslinked protein matrix being treated with a polar organic solvent.
 17. A method of making a protein hydrogel, the method comprising the steps of: (a) treating a protein with an acylating agent to yield an acylated protein; (b) crosslinking the acylated protein with a bifunctional crosslinking agent to yield a crosslinked protein; and (c) treating the crosslinked protein with a polar organic solvent.
 18. The method according to claim 17, wherein in step (a) the protein is treated with an amount of acylating agent sufficient to acylate from about 1 to about 98% of lysine residues within the protein.
 19. The method according to claim 17, wherein in step (a) the protein is treated with an anhydride.
 20. The method according to claim 17, wherein in step (a) the protein is treated with a tetracarboxylic acid dianhydride.
 21. The method according to claim 20, wherein in step (a) the protein is treated with ethylenediaminetetraacetic acid dianhydride.
 22. The method according to claim 20, wherein in step (b) the acylated protein is crosslinked with a dialdehyde.
 23. The method according to claim 22, wherein in step (b) the dialdehyde is selected from the group consisting of OCH—(CH2)_(x)—CHO wherein X is an integer of from 2 to
 8. 24. The method according to claim 23, wherein in step (b) the dialdehyde is glutaraldehyde.
 25. The method according to claim 17, wherein: in step (a) the acylating agent is added to an aqueous protein solution of about pH 12, at a temperature of about 65 to 100° C. to yield the acylated protein; and in step (b) crosslinking is effected by addition of glutaraldehyde to an aqueous solution of the acylated protein of step (a) to yield a crosslinked protein; and in step (c) the crosslinked protein is treated with a polar organic solvent selected from the group consisting of C₁-C₄ alcohols, C₁-C₄ ketones, and C₁-C₄ aldehydes.
 26. The method according to claim 25, wherein in step (c) the crosslinked protein is treated with a polar organic solvent selected from the group consisting of ethanol, propanol, butanol, and acetone.
 27. The method according to claim 26, wherein in step (c) the crosslinked protein is treated with ethanol. 